Lmx1a and lmx1b function cooperatively to regulate proliferation, specification, and differentiation of midbrain dopaminergic progenitors

Abstract

LIM homeodomain transcription factors, Lmx1a and Lmx1b, are required for the development of midbrain dopaminergic (mDA) neurons. Lmx1b is required for the specification and maintenance of mDA neurons, primarily due to its role in isthmic organizer development that is essential for the induction of mDA neurons. Here, we conditionally deleted Lmx1b in the ventral neural tube using ShhCre and found that Lmx1b conditional mutant mouse embryos show no defect in the development and maintenance of mDA neurons. In addition, Dreher (Lmx1a mutant) embryos display only a moderate reduction in the number of mDA neurons, suggesting that the related family member Lmx1b might compensate for Lmx1a function. We therefore generated Lmx1a and Lmx1b double mutants. Severe loss of mDA neurons occurred in Lmx1a(dr/dr);Shh(Cre/+);Lmx1b(f/f) double mutants due to essential roles for Lmx1a and Lmx1b in regulating the proliferation and neuronal commitment of mDA progenitors through the expression of Wnt1 and Ngn2, respectively. Lmx1a and Lmx1b also negatively regulate Hes1 expression and consequently cell cycle exit through activation of p27(Kip1) expression. In addition, Lmx1a and Lmx1b also regulate the expression of floor plate genes such as Corin and Slit2 and specification of postmitotic mDA neurons. These defects were more severe with decreasing gene dosage of Lmx1a and Lmx1b or observed only when all four copies of Lmx1a and Lmx1b genes were inactivated. Together, our results demonstrate that Lmx1a and Lmx1b function cooperatively to regulate proliferation, specification, and differentiation of mDA progenitors, including their floor plate-like properties.

Figure 1.

Lmx1b is deleted in the majority of the mDA domain before the generation of mDA neurons. A, C, E, G, I, K, Lmx1b transcripts were detected in the ventral midline of wild-type embryos in the midbrain. B, D, F, H, J, L, Lmx1b transcripts were only detected as two lateral stripes in the caudal midbrain and was undetectable in the rostral midbrain of Shh^Cre/+;Lmx1b^f//f embryos at E9.5, E10.5, and E12.5. Fgf signaling from the isthmus organizer is not disrupted in Shh^Cre/+;Lmx1b^f/f embryos as shown by whole-mount in situ hybridization for Fgf8 and for its downstream target Erm at E10.5 (M–P). The arrow in M and N indicates the position of the isthmus. Scale bars, 100 μm.

Figure 2.

Lmx1b is not required for mDA neuron development. A, B, Shh^Cre/+;Lmx1b^f/f embryos expressed mDA progenitor marker Lmx1a and mature mDA neuronal marker TH. C, There is no statistically significant difference in the number of TH+ cells in the whole ventral midbrain and caudal diencephalon region between wild-type and Shh^Cre/+;Lmx1b^f/f embryos. Error bars indicate SEM. Postmitotic mDA markers Lmx1b, Nurr1 (D, H), Pitx3 (E, I), AADC (F, J), and VMAT (G, K) were all normally expressed in Shh^Cre/+;Lmx1b^f/f embryos. Note that antibodies used to detect Lmx1a and Lmx1b also recognize mutated version of these proteins. Scale bars, 100 μm.

Figure 3.

Lmx1a and Lmx1b cooperatively regulate mDA neurons development. A, Quantification of mDA progenitors (B–F, Lmx1a+Ki67+), and immature (G–K, Nurr1+TH−) and mature (G–K, Nurr1+TH+) neurons in the whole midbrain region of Lmx1a/b single and compound mutants at E12.5. The expression of the specific mDA neuron marker Pitx3 is shown in L–P. The dashed line in the schematic embryo indicates where the sections are taken from in the rostrocaudal axis. *p < 0.05; **p < 0.01; ***p < 0.001; N.S., not significant. Error bars indicate SEM. Scale bar, 100 μm.

Figure 4.

Loss and incomplete differentiation of mDA neurons occurs in the ventral midbrain of Lmx1a/b double homozygous mutant embryos at E18.5. Expression of mature mDA neuron markers TH in combination with Nurr1 (A–E), Pitx3 (F–J), and Lmx1b (K–O) in E18.5 middle midbrain coronal sections from Lmx1a/b single and compound mutants. The few remaining TH+, Nurr1+ (D), TH+, Pitx3+ (I), or TH+, Lmx1b+ (N) mDA neurons in Lmx1a/b double mutants may be generated from caudal progenitors that did not undergo deletion of Lmx1b. Scale bars, 100 μm. D, Dorsal; L, lateral.

Figure 5.

Lmx1a/b are required for the proliferation of mDA progenitors in a gene dosage-dependent manner. Proliferation index is measured in Lmx1a/b single and compound mutants by quantification of S-phase progenitors (BrdU+/Ki67+) in the Lmx1a+ domain of whole midbrain and caudal diencephalon at E10.5 (K). A–E, mDA progenitors in active cell cycle expressed both Lmx1a and Ki67. F–J, mDA progenitor cells in S-phase of the cell cycle are double positive for Lmx1a and BrdU. Lmx1a+ region is demarcated by dotted lines. L–P, No change in apoptosis as measured by phosphorylated Caspase 3 staining. The dotted lines indicate the outline of the neural tube. *p < 0.05; **p < 0.01; N.S., not significant. Error bars indicate SEM. Scale bars, 100 μm.

Figure 6.

Cell cycle progression of mDA progenitors is inhibited in Lmx1a single and Lmx1a/b compound mutants at E10.5. The number of Cyclin D2-positive mDA progenitors is reduced in Lmx1a single and Lmx1a/b compound mutants (A–E, K). The cyclin-dependent kinase inhibitor p27^Kip1 is upregulated (F–J) and Wnt1 expression is lost in Lmx1a/b double mutants (M–Q). The graph in K shows the percentage of Cyclin D2+ cells in mDA domain delineated by Lmx1b staining (A–E, dotted lines). Quitting fraction (Qf) in the graph in L is obtained after a BrdU pulse at E10.5 and counting the fraction of BrdU-labeled cells at E11.5 that have exited the cell cycle (Brdu+Ki67−Lmx1b+/BrdU+Ki67+Lmx1b+). *p < 0.05; **p < 0.01; ***p < 0.001; N.S., not significant. Error bars indicate SEM. Scale bars, 100 μm.

Figure 7.

Lmx1a/b cooperatively regulate mDA progenitor specification. Msx1 expression is reduced and lost in Lmx1a^dr/dr;Shh^Cre/+;Lmx1b^f/+ and Lmx1a^dr/dr;Shh^Cre/+;Lmx1b^f/f mDA progenitor, respectively, at E12.5 (A–E). Lmx1b failed to be downregulated in Lmx1a^dr/dr;Shh^Cre/+;Lmx1b^f/f mutant (I), whereas Sox2 expression appears normal (F–J). Nkx6.1 expression remains unchanged in all mutant embryos (K–O). The dotted lines correspond to mDA domain delimited by Lmx1a. Scale bars, 100 μm.

Figure 8.

Lmx1a/b regulate midbrain floor plate identity. Expression of floor plate markers, Arx (A–E), Corin (F–J), Slit2 (K–O), and Shh (P–T) in midbrain coronal sections at E12.5. The arrowheads indicate dorsal position of floor plate in P–T based on the dorsal limit of Arx expression in corresponding A–E. ImageJ software is used to quantify Shh expression between these two regions. Expression ratio is obtained by dividing the mean intensity value from boxed area a by the mean intensity value in boxed area b. Note that Shh expression in S is high in both basal and floor plate progenitors. Scale bar, 100 μm.

Figure 9.

Lmx1a/b regulate neurogenesis of mDA progenitors. Ngn2 and Mash1 expression are reduced in Lmx1a^dr/dr;Shh^Cre/+;Lmx1b^f/+ and Lmx1a^dr/dr;Shh^Cre/+;Lmx1b^f/f mutant embryos at E12.5 (A–K), as well as the expression of Dll1 (L–P), a direct target of Ngn2. Nato3 expression is unchanged (Q–U), but Hes1 is upregulated in Lmx1a^dr/dr;Shh^Cre/+;Lmx1b^f/+ and Lmx1a^dr/dr;Shh^Cre/+;Lmx1b^f/f mutant embryos (V–Z). Cell counts in the graph in K are done by calculating the fraction of Ngn2+Ki67+ double-positive cells over the Ki67+ positive cells within the mDA progenitor domain delineated by Lmx1a-stained cells. Data are represented as percentage of wild-type littermate numbers. *p < 0.05; **p < 0.01. Error bars indicate SEM. Scale bars, 100 μm.

Figure 10.

Lmx1a/b are required for the specification of postmitotic mDA precursors. Scattered Lmx1b+ neurons misexpressing the red nucleus neuron markers Lim1/2 (A–E, A′–E′) and Brn3a (K–O, K′–O′) are found at the margin of mDA domain in Lmx1a single and Lmx1a/b compound mutant embryos at E12.5. The oculomotor neuron marker Isl-1 does not colocalizes with Lmx1b+ neurons in any Lmx1a/b mutants (F–J, F′–J′). Zones delimited by the white squares correspond to the high magnification in A′–O′. The white arrowheads indicate double-labeled neurons. Scale bars: A, 100 μm; A′, 500 μm.